Sphingosine-1-phosphate (Si P), a potent lipid mediator produced from sphingosine by sphingosine kinases (SphKs), regulates many processes important for cancer progression, including cell growth and survival (Spiegel et al., Nature Rev Mol Cell Biol. 4:397-407, 2003). In contrast to S1P, its precursors, sphingosine and ceramide, are associated with growth arrest and induction of apoptosis (Ogretman & Hannun, Nature Rev Cancer 4:604-616, 2004). Thus, the balance between these interconvertible sphingolipid metabolites has been viewed as a cellular rheostat determining cell fate (Cuvillier et al., Nature 381:800-803, 1996). Numerous studies have shown that perturbations in the S1P/ceramide rheostat are involved in the regulation of resistance to chemotherapy and radiation therapy of neoplastic cells, including those of hematopoietic origin (Ogretman et al., supra.; Hait et al., Biochim Biophys Acta 1758:2016-2026. 2006; and Milstien & Spiegel, Cancer Cell 9:148-150, 2006).
Two Sphk isoenzymes, SphKI and SphK2, have been described which, while sharing many features (Kohama et al., J. Biol Chem 273:23722-23728, 1998; and Liu et al., J. Biol Chem 275:19513-19520, 2000) exhibit distinct functions. SphK1 promotes cell growth and survival (Olivera et al., J Cell Biol 147:545-558, 1999; Xia et al., J. Biol Chem 277:7996-8003, 2002; Bonhoure et al., Leukemia 20:95-102, 2006; and Sukocheva et al., J Cell Biol 173:301-310, 2006), whereas SphK2, when overexpressed, has opposite effects (Maceyka et al., J Biol Chem 280:37118-37129, 2005; and Okada et al., J Biol Chem 280:36318-36325, 2005). SphK1 is a key enzyme that regulates the S1P/ceramide rheostat (Maceyka et al., supra.; Berdyshev et al., Cell Signal 18:1779-1792, 2006; and Taha et al., FASEB J 20:482484, 2006). Indeed, S1P and SphK1 have long been implicated in resistance of both primary leukemic cells and leukemia cell lines to apoptosis induced by commonly used cytotoxic agents (Cuvillier et al., Nature, 2004 supra.; Cuvillier et al., J. Biol Chem 273:2910-2916, 1998; Cuvillier et al., Blood 98:2828-2836, 2001; and Jendiroba et al., Leuk Res 26:301-310, 2002). Non-isozyme specific inhibitors of SphKs, such as L-threo-dihydrosphingosine (safingol) and N,N-dimethylsphingosine (DMS), are cytotoxic to leukemia cells (Jarvis et al., Mol Pharmacol 54:844-856, 1998; and Jendiroba et al., 2002, supra.). Interestingly, multi-drug resistant HL-60 myelogenous leukemia cells were more sensitive to DMS than the parental cells (Jendiroba et al., 2002, supra.). Moreover, SphK1 activity was lower in HL-60 cells sensitive to doxorubicin or etoposide than in MDRI- or MRP1-positive HL-60 cells. Enforced expression of SphKI in sensitive HL-60 cells blocked apoptosis whereas downregulation of Sphk1 overcame chemoresistance by inducing mitochondria-dependent apoptosis (Bonhoure et al., 2006, supra.). These observations take on added significance in light of evidence that MDR expression is a strong prognostic indicator in acute myelogenous leukemia (AML) (Filipits et al., Leukemia 14:68-76, 2000) and that the MDR phenotype, which commonly arises following treatment of AML with anthracyclines or plant-based alkaloids, is thought to represent an obstacle to successful chemotherapy. In addition, resistance of K562 human chronic myeloid leukemia cells to Imatinib, an inhibitor of Bcr-Abl tyrosine kinase, correlated with expression of SphK1 and generation of S1P, whereas downregulation of SphK1 increased sensitivity to Imatinib-induced apoptosis in resistant cells (Baran et al., J Biol Chem 282:10922-10934, 2007). Thus, the development of effective and specific inhibitors of SphK1 might prove useful not only in diminishing levels of pro-survival S1P, but also in potentiating ceramide generation, a process that mediates, at least in part, the pro-apoptotic actions of certain cytotoxic agents (Maggio et al., Cancer Res 64:2590-2600, 2004; Rahmani et al., Cancer Res 65:2422-2432, 2005; and Rosato et al., Mol Pharmacol 69:216-225, 2006).
Sphingosine kinase inhibitors have been described (Kim et al., Bioorg & Med Chem 13:3475-3485, 2005; Kono et al., J. Antibiotics 53:459-466, 2000; Kono et al., J. Antibiotics 53:753-758, 2000; Marsolais & Rosen, Nature Reviews/Drug Discovery 8:297-307, 2009; and US 2008/0167352 A1 (Smith et al., published Jul. 10, 2008). None of these publications describe, however, the novel sphingosine kinase Type 1 inhibitors herein.
Here we describe a potent, water-soluble inhibitor of SphK1 (SK1-I) that triggers multiple perturbations in activation of various signaling and survival-related proteins. Sk1-I markedly induced apoptosis in human leukemic cell lines as well as blasts obtained from patients with AML and inhibited growth of AML xenograft tumors. Sk1-I serves as model for other related compounds which are described further below.